MP currently utilizes two types of animal models: animals that escalate their alcohol intake following a prolonged history of intermittent brain alcohol exposure (post-dependent), or animals that have interesting alcohol-related phenotypes as a function of genetic factors, such as rats selectively bred for high alcohol preference, or mice or rats that have been genetically engineered. These different models may tap into different biological mechanisms, and are therefore complementary. Voluntary alcohol consumption and its escalation over time are studied in several models. Relapse-like behavior is studied in animals using reinstatement models, in which lever-pressing for alcohol is first established, and then extinguished by removing the alcohol. Presentation of drug-associated cues or exposure to stress will lead to resumption of lever-pressing on the previously alcohol-delivering lever (reinstatement). Reinstatement induced by cue or stress is differentially sensitive to pharmacological manipulations, and evaluating candidate drugs for their ability to suppress reinstatement induced by the two different types of stimuli guides optimal selection of target populations in initial clinical trials. Information may also be gained on how treatments might be combined to obtain additive effects. Mu-opioid receptor gene (OPRM1) The opioid receptor antagonist naltrexone (NTX) is an established treatment for alcoholism, however there is considerable heterogeneity of responses in clinical populations. Recent evidence suggests that a non-synonymous SNP, 118A&#8594;G, found in the human OPRM1 gene that encodes the mu-opioid receptor may modulate alcohol reward and therapeutic response to NTX. In an attempt to establish the mechanistic role of human OPRM1 A118G variation, and to isolate the effects of this polymorphism from those of alleles in linkage disequilibrium with it, we created two lines of humanized mice carrying the respective human allele (118AA and 118GG). We observed a markedly enhanced dopamine (DA) release in the nucleus accumbens of 118GG mice in response to alcohol. This finding was consistent with a positron emission tomography study conducted in parallel in humans, which found a more vigorous DA response to alcohol in the ventral striatum in 118G carriers (Ramchandani et al., 2011). Further work with the humanized mouse lines revealed elevated alcohol consumption in 118GG mice, and experiments are underway to investigate whether 118GG mice will be preferentially or selectively sensitive to NTX (Thorsell et al., in preparation). Substance P and its NK1 receptor Substance P mediates stress responses, and blockade of the receptor for substance P, NK1R, produces anti-stress effects. We have shown that genetic deletion of NK1R in mice leads to markedly decreased alcohol preference, a lack of conditioned place preference for alcohol, and an absence of escalated intake over time. Furthermore, pharmacological blockade of NK1 receptors using the NK1 antagonist L-703,606 suppresses alcohol consumption similar to genetic deletion of the receptor (Thorsell et al., 2010). We recently collaborated with Dr. Kenner Rice to resynthesize a literature compound, L-822429, with high affinity for the rat NK1 receptor. L-822429 dose-dependently suppressed stress-induced reinstatement of alcohol seeking in Wistar rats, but had no effect on cue-induced reinstatement, suggesting the possibility of additive effects of an NK1R antagonist and opioid antagonists such naltrexone (Schank et al., in press). In follow-up work using genetically selected alcohol preferring P-rats, L-822429 dose-dependently suppressed self-administration rates in P-rats but was ineffective in non-dependent Wistar rats. A nuclease protection assay showed an innate upregulation of NK1R receptor expression in several brain regions in P rats, with the greatest up-regulation within the amygdala (Schank et al., in preparation). Together, these finding further support NK1R antagonism as a candidate target for the treatment of alcoholism. Corticotropin-Releasing Hormone (CRH) and its CRH1 receptor Elevated self-administration of alcohol and an increased behavioral sensitivity to stress in the post-dependent state is in large part mediated by an up-regulation of the CRH1 subtype of CRH receptors in the amygdala (Sommer et al., 2008). We had previously identified a series of CRH antagonists with suitable properties for clinical development (Gehlert et al., 2007;Thorsell et al., unpublished data). Two of the candidates, pexacerfont and GSK561679, were selected to enter experimental medicine studies in our clinical program under CRADAs with Bristol Myers Squibb and GlaxoSmithKline respectively. Those studies are currently underway. Neuropeptide Y (NPY) NPY is a an endogenous anti-stress peptide that counteracts the behavioral stress effects of CRH. Activation of NPY-Y1 receptors, or blockade of presynaptic Y2 autoreceptors, suppresses escalated post-dependent drinking, while leaving basal intake of alcohol in non-dependent animals unaffected (reviewed in Thorsell et al., 2006). NPY also potently suppresses reinstatement of alcohol seeking induced by the pharmacological stressor yohimbine (Cippitelli et al., 2010). Because orally available, brain penetrant Y1 agonists are currently unavailable, we evaluated a non-peptide compound under a CRADA with Johnson and Johnson Pharmaceuticals, JNJ-31020028, targeting the Y2 receptor to determine its potential for clinical development. Although showing some of the expected activity, the effects of JNJ-31020028 followed an inverted-U-shaped dose-response curve, possibly reflecting activity at Y2 heteroceptors on glutamatergic terminals at higher doses (Cippitelli et al., 2011). Thus, despite the scientific appeal of targeting the NPY system, this line of research is no longer of high priority in our program. Neuropeptide S (NPS) NPS suppresses anxiety and appetite in experimental animals, and initial work in collaboration with the Ciccocioppo laboratory indicated that NPS signaling may play a role in relapse to alcohol seeking (Canella et al., 2009). We developed a screenable assay for the NPS receptor, and in collaboration with the NIH Chemical Genomics Center (NCGC) identified a lead molecule, NCG001865684, that was determined to be brain penetrant upon peripheral administration in rats. Pretreatment with NCG001865684 was found to prevents NPS-induced suppression of feeding, and to suppress operant responding for alcohol. However, no effect was found on cue- or stress-induced reinstatement of alcohol seeking. Optimization is currently ongoing at NCGC, and is focused on engineering out off-target activity discovered in the course of a standard CEREP screen of common drug targets. Gene expression profiling and epigenetic modifications A series of projects are being carried out looking at changes in gene expression and epigenetic modifications in the post-dependent rat model to identify potential pharmacological targets. Bioinformatic analysis of the initial runs applying RNAseq, CHIPseq and microRNA (miR) has identified several interesting, differentially expressed transcripts as well as several cases of miR-mediated changes in gene expression (Tapocik et al., in preparation). Investigation of epigenetic modifications revealed increased DNA methyltransferase activity in the hippocampus of post-dependent rats, and hypermethylation induced in the brain by L-methionine injection was found to increase alcohol self-administration as well as the motivation to consume alcohol (Barbier et al., in preparation).